Oligopeptide microarrays are widely used in research and healthcare. Within these areas, oligopeptide microarrays are suitable for many different applications. Oligopeptide microarrays for example provide a tool for the identification of biologically active motifs, e.g. oligopeptide microarrays may imitate potential active motifs of ligands for screening the binding to corresponding receptors. Furthermore, the oligopeptide microarrays might reflect specific sequences of disease associated antigens. Such oligopeptide microarrays can be utilized to detect antibodies from patient samples suggesting the presence of certain inflammatory diseases, infections, and the like. Another important application of the oligopeptide microarrays is the discovery of biochemical interactions, including the binding of proteins or DNA. Oligopeptide microarrays can further be used for the profiling of cellular activity, the activity of enzymes, the adhesion of cells, and the like.
Traditional methods for the analysis of autoimmune diseases involves the detection of autoantibodies and include enzyme linked immunosorbent assays (ELISAs), Western blot analysis, immunoprecipitation analysis and flow-based assays. Routine assays for detection of autoantibodies is generally performed by ELISAs and fluorescence assays. Individual assays are performed in microtiter plates, with a single antigen per well. These tests are performed one-at-a-time, are laborious, and expensive. Oligopeptide arrays have been used to characterize and detect autoantibodies, but they have generally utilized purified antigen molecules spotted onto substrates. The antigens must be produced in recombinant expression systems and purified, which is a time-consuming process. These antigens are generally whole proteins, or known antigenic domains, and do not allow the characterization of specific epitopes. Synthetic peptide arrays have been utilized as well, however the production of these peptides is done by commercial automated peptide synthesizers, and then spotted onto slides. However, they cannot achieve the scale of peptides synthesized by maskless array synthesis (MAS) technology.
Traditional methods, such as ELISA, are laborious and costly, and can only be done one antigen at a time. While spotted oligopeptide microarrays are available, and allow parallel detection of multiple autoantibodies, the cost of producing those arrays is very expensive due to the cost of producing purified antigen molecules in a recombinant expression system. In addition they have a very low resolution and cannot achieve the comprehensive coverage of substantially the whole proteome that an oligopeptide microarray can. Many proteins cannot be synthesized in in vitro systems, which would prevent their use on such arrays.
Furthermore, antigens that are expressed and then spotted onto a microarray often only represent a small percentage of the full protein sequence. Antibodies in one patient may target one set of antigenic domains, which in another patient, the antibodies may target a completely different set of antigenic domains in the same protein. Such patient-to-patient differences could arise from misfolding of proteins, a common problem in autoimmune disease, thus causing differential presentation of protein domains to antibody producing B cells. Oligopeptide arrays are thus preferred because they allow all possible antigenic sites within a given protein to be examined in order to detect patterns or fingerprints across many patients.
The object of the present invention is the provision of microarrays with a high density oligopeptides with improved capabilities for high resolution analysis (including, but not limited to, serological analysis), a method for their synthesis and their use. The advantage of the microarrays according to the invention is their oligopeptide density and the coverage of substantially the whole proteome of an organism by the application of a tiling concept. Because of this oligopeptide density, the microarrays according to the invention allow the parallel detection of all autoantibodies in a human serum sample with a single binding assay. In addition, specific information about the location of epitopes is obtained from the assay by the introduction of the tiling concept. Therefore, the present invention provides a simple, cost-effective method for screening for a wide variety of autoimmune diseases, as well as rapid custom epitope mapping, screening peptides for small molecule binding, synthesis of antibody-like arrays for protein expression analysis, proteome-scale peptide scanning, and many more applications.